Phage antibodies

ABSTRACT

Peripheral blood leucocytes incubated with a semi-synthetic phage antibody library and fluorochrome-labeled CD3 and CD20 antibodies were used to isolate human single chain Fv antibodies specific for subsets of blood leucocytes by flow cytometry. Isolated phage antibodies showed exclusive binding to the subpopulation used for selection or displayed additional binding to a restricted population of other cells in the mixture. At least two phage antibodies appeared to display hithereto unknown staining patterns of B lineage cells. This approach provides a subtractive procedure to rapidly obtain human antibodies against known and novel surface antigens in their native configuration, expressed on phenotypically defined subpopulations of cells. Importantly, this approach does not depend on immunization procedures or the necessity to repeatedly construct phage antibody libraries.

This application is a continuation of application Ser. No. 09/085,072filed May 26, 1998, now U.S. Pat. No. 6,265,150, which is a continuationin part of application Ser. No. 08/932,892 filed Sep. 18, 1997, nowabandoned, which is a File-Wrapper-Continuation of Ser. No. 08/483,633filed Jun. 7, 1995, now abandoned, both of which are incorporated hereinby reference.

BACKGROUND OF THE INVENTION

The construction of libraries of fragments of antibody molecules thatare expressed on the surface of filamentous bacteriophage and theselection of phage antibodies (Phabs) by binding to antigens have beenrecognized as powerful means of generating new tools for research andclinical applications. This technology, however, has been mainly used togenerate Phabs specific for purified antigens that are available insufficient quantities of solid-phase dependent selection procedures. Theeffectiveness of such Phabs in biochemical and functional assays varies;typically, the procedure used to select Phabs determines their utility.

Typically, many antigens of interest are not available in pure form invery large quantities. This clearly limits the utility of Phabs inbinding such materials for research and clinical applications. Further,the utility of Phabs in such applications is directly proportional tothe purity of the antigens and purification methods to assure thespecificity of the isolate Phabs. Human monoclonal antibodies that bindto native cell surface structures are expected to have broad applicationin therapeutic and diagnostic procedures. An important extension ofphage antibody display technology would be a strategy for the directselection of specific antibodies against antigens expressed on thesurface of subpopulations of cells present in a heterogenous mixture.Ideally, such antibodies would be derived from a single highly-diverselibrary containing virtually every conceivable antibody specificity.

SUMMARY OF INVENTION

A library was constructed from 49 human germline V_(H) genes fused to aJ_(H)4 gene and partly randomized CDR3 regions varying in length between6 and 15 amino acids. The CDR3 regions were designated to contain shortstretches of fully randomized amino acid residues flanked by regions oflimited variability. Residues in the latter portion of CDR3 wereselected based on their frequent occurrence in CDRs(complementarity-determining regions) of natural antibody molecules,random CDR3 with an increased frequency of clones producing functionalantigen binding sites. The synthetic V_(H) segments were combined withseven different V_(L) genes and expressed as geneIII-scFv fragments onthe surface of phage, resulting in a library of 3.6×10⁸ clones. Thislibrary was used to isolate monoclonal phage antibodies (MoPhabs) to avariety of different structures (haptens, proteins and polysaccharides)by selection on solid phase-bound antigen.

Further, MoPhabs were also isolated by flow cytometry, resulting inMoPhabs specific for subpopulations of cells present in a heterogenousmixture. These antibodies detect known and novel structures on variouspopulations of blood and fetal bone marrow cells.

DETAILED DESCRIPTION OF INVENTION

The phage antibodies of the instant invention are obtained from alibrary of phage antibodies which possess specificity for a plurality ofantigens. In practice, such libraries can be obtained from a variety ofsources or constructed by known methods. A method particularly usefulfor constructing such libraries is described in paper by G. Winter, etal., Annual Reviews of Immunology, 12, 433-455 (1994), which isincorporated by references.

The library is then admixed with the antigens (as used herein, antigenshall be inclusive of haptens and antigen analogs) of interest and thephage antibodies bound to these antigens are then isolated. Theprocedure may be repeated until a population of phage antibodies havingthe desired specificity(ies) is obtained, and the isolated phageantibodies may then be cloned by conventional methods known to those inthe art.

In a preferred embodiment, the phage antibody library is admixed with acell mixture labeled with a fluorescent labeled antigen, or a pluralityof antigens each labeled with a different fluorescent label, and sortedby flow cytometry. Preferred labels include phycoerythrin (PE), PerCP,and fluorescein isothiocyanate (FITC). The phage antibodies bound tocells, thus obtained, can be eluted. The phage antibodies (phages thatexpress antibody specificities of interest) can then be cloned byconventional techniques to obtain a plurality of phage antibodies havinghigh specificity for single antigens.

EXAMPLES

The following examples illustrate certain preferred embodiments of theinstant invention, but are not intended to be illustrative of allembodiments.

Example 1

Library Construction

The semi-synthetic Phab library was constructed essentially as describedin Hoogenboom and Winter, J. Mol. Biol. 227, 381-388 (1992) and Nissimet al. EMBO 13,692-698 (1994). Briefly, degenerate oligonucleotides wereused to add synthetic CDR3 regions to a collection of 49 previouslycloned germline V_(H) genes. Subsequently, these in vitro ‘rearranged’V_(H) genes were cloned into a collection of pHENI phagemid-derivedvectors containing 7 different light chain V regions, fused in frame tothe gene encoding the phage minor capsid protein geneIII. Introductionof these constructs into bacteria results, in the presence of helperphage, in the expression of scFv antibody fragments as geneIII fusionproteins on the surface of bacteriophage.

Plasmid DNA containing the V_(κ)3 gene expressed in EBV-transformed cellline was amplified with primers V_(κ)3LINK and J_(κ)4B to introduce Ncoland Xhol restriction sites and the (G4S) linker sequence. Amplifiedproduct was cloned into the pHEN1 phagemid vector using NcoI and XhoIresulting in pHEN1-V_(κ)3. Total RNA was isolated from fetal bone marrowB lymphocytes, converted to cDNA by oligo-dT priming and amplified byPCR using V_(κ)1, V_(κ)2, V_(κ)4, V_(λ)1 and V_(λ)2 gene family-specificprimers. All PCR reactions were carried out in a volume of 50 μl with250 μM dNTPs, 20 pmol of each primer and 0.2 units of Taq DNA polymerase(Supertaq, HT biotechnology Ltd. Cambridge, UK) in the manufacturerrecommended buffer. PCR reactions consisted of 25 cycles of 1 minute at94° C., 1 minute at 58° C. and 2 minutes at 72° C.). PCR amplifiedproducts were digested with SacI and NotI and ligated in thepHEN1-V_(κ)3 vector digested with the same enzymes. This resulted in theconstruction of 7 pHEN 1-derived vectors, each containing a rearrangedmember of the V_(κ)1, V_(κ)2, V_(κ)3, V_(κ)4, V_(λ)1, V_(λ)2 and V_(λ)3gene families, the scFv linker and restriction sites XhoI and NcoI forcloning of the heavy chain library. Nucleotide sequences of the V_(L)genes appear in the EMBL, Genbank and DDBJ Nucleotide Sequence Databasesunder accession numbers X83616 and X83712-X83714.

PCR primers were designed to fuse a bank of 49 germline V_(H) genes(Tomlinson et al., J. Mol. Biol. 227, 776-798 (1992)) to CDR3 regions,varying in length from 6 to 15 residues, and a J_(H)4 gene segment.Template, consisting of 0.5 ng of a mixture of plasmids encoding genesfrom a single V_(H) gene family, was amplified using the V_(H) familybased primers VHBackSfi (Marks et al., J. Mol. Biol. 222, 581-597(1991)) and one of the CDR3 primers. PCR products of each amplificationencoding a differently-sized HCDR3 loop were digested with XhoI and NcoIand cloned into the pHEN1-V_(λ)1 vector. This resulted in a phagemidlibrary of 1.2×10⁸ clones. Plasmid DNA from this library was digestedwith XhoI-NcoI and the synthetic V_(H) regions were cloned into theother pHENI-light chain vectors, resulting in seven libraries, eachvarying in size between 2×10⁷ and 1.2×10⁸ clones. The seven librarieswere rescued individually (Marks et al., EMBO 12, 725-734 (1993)) usinghelper phage VCS-M13 (Stratagene) and finally combined to form a singlelibrary of 3.6×10⁸ clones.

Example 2

Selection of Phage Antibodies

The phages were panned for binding to antigen-coated immunotubes (NuncMaxisorp; Marks et al. J. Mol. Biol. 222, 581-597 (1991) using thefollowing antigens: dinitrophenol (DNP) coupled to BSA, tetanus toxoid(TTX), tyraminated Group B Streptococcal type III capsularpolysaccharide (GBS), human surfactant protein A (spA; Hawgood,Pulmonary Surfactant. From Molecular Biology to Clinical Practice.Elsevier Science Publishers, pp. 33-54 (1992), human thyroglobulin (Tg;Logtenberg et al., J. Immunol. 136,1236-1240 (1986)), human VonWillebrand Factor (VWF), human VWF fragment A2, a purified human IgGparaprotein, a recombinant protein corresponding to the HMG domain of Tcell-specific transcription factor TCF-1 (HMG, van Houte et al, J. Biol.Chem. 268, 18083 (1993), a deletion mutant of the epithelialglycoprotein EGP-2 (δEGP-2; Helfrich et al., Int. J. Cancer, Suppl. 8,1.(1994), the extracellular portion of human ICAM-1, (Hippenmeyer et al.Bio. Technology 11, 1037 (1993), an uncharacterized DNA binding proteinisolated from a cDNA library and expressed as a maltose binding protein(MBP) fusion protein (BLT1/MBP), and the human homeobox protein PBXla(Monica et al. Mol. Cell. Biol. 11, 6149-6157 (1991). All antigens werecoated overnight at room temperature at a concentration of 10 ug/ml inPBS (DNP-BSA, GBS, Tg, VW, A2, TTX, ICAM-1, BLT1/MBP, PBX1a) or 50 mMNaHCO₃ pH 9.6 (IgG, spA, HMG, δEGP-2).

To target selection of Phabs to a desired portion of a molecule, phageselections were performed on solid phase-bound BLT1/MBP fusion proteinas described in the standard protocol with the addition of 6 μg/mlsoluble MBP to the Phab-milkpowder mixture during panning. In order toobtain Phabs capable of discriminating between two highly homologousproteins, selections on immunotube-coated full-length PBX1a were carriedout according to the standard protocol in the presence of 5 μg/mlfull-length recombinant PBX2 protein during panning (Monica et al., Mol.Cell. Biol 11, 6149-6157 (1991).

Example 3

Selection of Phage Antibodies by Cell Sorting

Venous blood was diluted 1:10 in 0.8% NH₄Cl/0.08% NaHCO₃/0.08% EDTA (pH6.8) to remove erythrocytes and the nucleated cells were pelleted andwashed once in PBS/1% BSA. Approximately 10¹³ phage antibody particleswere blocked for 15 minutes in 4 ml 4% milkpowder in PBS (MPBS). 5×10⁶leucocytes were added to the blocked phages and the mixture was slowlyrotated overnight at 4° C. The following day, cells were washed twice in50 ml ice-cold PBS/1% BSA. The pelleted cells were resuspended in 50 μlof CD3-PerCP and 50 μl of CD20-FITC and after a 20 minute incubation onice, cells were washed once with 1% BSA/PBS and resuspended in 500 μlice-cold PBS/1% BSA. Cell sorting was performed on a FACSvantage®. Foreach subpopulation, 10⁴ cells were sorted in 100 μl PBS.

Example 4

Propagation of Selected Phases

Phages were eluted from the cells by adding 150 μl 76 mM citric acid pH2.5 in PBS and incubation for 5 minutes at room temperature (RT). Themixture was neutralized with 200 μl 1 M Tris/HCl, pH 7.4. Eluted phageswere used to infect E'Coli X11-Blue and the bacteria were plated on TYEmedium containing the appropriate antibiotics and glucose. Bacterialcolonies were counted, scraped from the plates and used as an inoculumfor the next round of phage rescue.

Example 5

Preparation of Monoclonal Phage Antibodies and scFy Fragments andImmunofluorescent Analysis

Phages were prepared from individual ampicillin resistant colonies grownin 25 ml 2TY medium, purified by polyethylene glycol precipitation,resuspended in 2 ml PBS, filtered (0.45 μM) and stored at 4° C. untilfurther use. ScFv fragments were produced in E Coli non-suppressorstrain SF 110 that is deficient in the proteases degP and ompT. In ourexperience, the stability of scFv produced in SF110 is superior to thatof scFv produced in HB2151 commonly used for this purpose.

For staining of leucocytes, 100 μl MoPhab was blocked by adding 50 μl 4%MPBS for 15 minutes at RT. 5×10⁵ leucocytes in 50 μl PBS/1% BSA wereadded and incubated on ice for 1 hour. The cells were washed twice inice-cold PBS/1% BSA. To detect cell-bound phages, the cells wereincubated in 10 μl of {fraction (1/200)} diluted sheep anti-M13polyclonal antibody (Pharmacia, Uppsala. Sweden), washed twice andincubated in 10 μl of 20 μg/ml PE-labeled donkey anti-sheep polyclonalantibody (Jackson Immunoresearch, West Grove, Pa.), each for 20 minuteson ice. The cells were washed and incubated in 10 μl each of CD3-FITCand CD20-PerCP monoclonal antibodies. When cells were strained withpurified scFv fragments, second and third step reagents consisted of theanti-myc tag-specific antibody 9E10 and FITC- or PE-labeled goatanti-mouse antibodies. After a single final wash, the cells wereresuspended in 0.5 ml PBS/1%BSA and analyzed by FACS.

Fetal bone marrow was from aborted fetuses (16-22 weeks gestation) andused following the guidelines of the institutional review board ofStanford Medical School Center on the use of human subjects in medicalresearch. Bone marrow cells were obtained by flushing intramedullarycavities of the femurs with RPMI 1640 medium. Pelleted cells weretreated with the hypotonic NH₄Cl solution to remove erythrocytes. 10⁶fetal bone marrow cells were stained with MoPhabs T1, B9, and B28 incombination with a panel of fluorochrome-labeled MoAbs. The panelincludes CD3 (Leu 4B PerCP), CD4 (Leu FITC), CD8 (Leu2a APC), CD 10(anti Calla FITC; all from Becton Dickinson Immunocytometry Systems, SanJose, Calif.), and FITC-conjugated goat anti-human, μ, δ, and κchain-specific polyclonal antibodies (Southern Biotechnologies, Ala.).

Example 6

Specificity of Isolated MoPhabs

5×10⁶ erythrocyte-lysed peripheral blood cells from a healthy individualwere incubated with the phage library and subsequently stained with CD3PerCP and CD20 FITC labeled monoclonal antibodies (MoAbs). Thepopulation was run on a flow cytometer.

10⁴ cells of each population were sorted and the phages bound to theisolated cells were eluted from the cell surface. The number of clonesobtained after the first round of selection varied between 320 and 1704.The number of phage clones obtained roughly was inversely correlatedwith the frequency of the cell population in the blood sample as shownin Table 1.

TABLE 1 # MoPhabs Sorted # Staining Population Round 1 Round 2 # Pos.Clones Profiles ‘all’ leucocytes  640  980 15/15 1 eosinophils 1280  39011/15 2 T-cells (CD3⁺)  320 3330 15/15 2 B-cells 1704 6000 10/16 3(CD20⁺)

The second round of selection resulted in a modest increase in thenumber of phages eluted from the cells in most but not all cases asshown in Table 1.

The phages eluted from the sorted cells were expanded as individuallibraries and used in a second round of selection employing the sameprocedure. Finally, MoPhabs were prepared from individual coloniesobtained after the second round of selection.

The binding properties of 15 MoPhabs from each sorted population wasanalyzed by incubation with peripheral blood leucocytes followed byincubation with secondary anti-phage PE-labeled antibody and CD20 FITCand CD3 PerCP. After two rounds of selection, between 63% and 100% ofthe MoPhabs were found to display binding activity to leucocytes, seeTable 1.

Staining profiles were obtained for a negative control MoPhab, a MoPhabderived from sorting ‘all’ leucocytes, two eosinophil-derived MoPhavs(E1/E2), two T cell derived MoPhabs 9T1fF2) and two B cell derivedMoPhabs (B9/B28). ScFv fragments were produced from each MoPhab clone.For all clones, identical results were obtained for whole phageantibodies and isolated scFv fragments, albeit some loss of signalintensity was observed when using the latter. The 15 MoPhAbs selected on‘all’ leukocytes showed identical staining patterns: all granulocytes,eosinophils, and monocytes stained homogeneously bright. All the Tlymphocytes stained but with varying intensity. Strikingly, no bindingto B lymphocytes was observed. Among the 15 MoPhAbs selected for bindingto eosinophils, two staining patterns were discernable. Both MoPhabsbound to all eosinophils and monocytes; the staining profile ofgranulocytes differed between both MoPhabs. MoPhab E2 reacted with themajority of T cells, whereas virtually no staining of T cells wasobserved with MoPhab E1. Conversely, MoPhab E2 did not bind to B cellswhile MoPhab E1 stained virtually all B cells. Two staining patternscould be distinguished among the 12 MoPhabs selected for binding to Tlymphocytes. MoPhab T2 dimly stained a subpopulation of B cells, T cellsand granulocytes but not monocytes and eosinophils. MoPhab T 1exclusively and brightly stained a subpopulation of T lymphocytescomprising approximately 50% of CD3⁺ cells. Finally, among MoPhabsselected from B cells, three staining patterns were distinguishable:approximately 50% of the peripheral blood B cells stained with MoPhabB9, MoPhab B28 stained all CD20⁺ peripheral blood B cells, whereasMoPhab B 11 stained virtually all leucocytes.

MoPhabs TI, B9 and B28 were selected for further characterization. Infour color staining experiments with CD3, CD4, CD8 and T 1 antibodies, T1 was shown to bind to CD8⁺ cells and not to CD4⁺ cells.Immunofluorscent staining of COS cells transiently transfected withcDNAs encoding the CD8α chain, the CD8β chain or both demonstrated thatMoPhAb T1 recognized cells expressing the CD8αα homodimer. We concludethat T1 recognizes an epitope encoded by the CD8α chain.

Triple-staining of B9 with CD20 and antisera specific for theimmunoglobulin μ, δ, γ, α, κ, and λ chains revealed that B9 markerexpression did not concur with any of the Ig isotypes. Triple-stainingof purified tonsil B cells with MoPhab B9 or B28, CD 19, and CD 10 or μheavy chain specific antibodies confirmed that B28 binds to all and B9binds to a subpopulation of CD 19⁺ tonsil B cells. Germinal center Bcells (CD19⁺/CD10⁺) uniformly lack the antigen recognized by MoPhab B9.In human bone marrow, the CD 19 marker is expressed from the earliestpro-B cell to the virgin, surface IgM⁺ B cell stage. Triple staining offetal bone marrow cells with CD 19, sIgM and B9 or B28 demonstrated thatB9 and B28 are not expressed during B lineage differentiation. Weconclude that the structures detected by the B9 and B28 MoPhabs areexpressed at a very late stage of B cell development, presumably afternewly generated sIgM+B cells have left the bone marrow. To our bestknowledge B cell-specific markers with such expression patterns have notbeen described previously.

Nucleotide sequence analysis was used to established V_(H) and V_(L)gene utilization and heavy chain CDR3 composition encoding the scFvantibodies obtained from the sorted subpopulations as shown in Table II.

TABLE II V_(H) and V_(L) gene utilization and deduced amino acidsequence of CDR3 regions of selected MoPhabs. MoPhab CDR3 V_(H) V_(L) A1RMRFPSY DP32 Vλ3 E1 RLRSPPL DP32 Vλ2 E2 RAWYTDSFDY DP45 Vκ1 T1KWLPPNFFDY DP32 Vκ3 T2 RSTLADYFDY DP69 Vλ3 B9 KGVSLRAFDY DP31 Vκ1 B28RGFLRFASSWFDY DP32 Vλ3

ScFv derived from different clones with the same staining profile showedidentical nucleotide sequences of CDR3 regions. The MoPhabs withdifferent staining patterns were encoded by various combinations ofV_(H) and V_(L) chains, with an overrepresentation of the DP32 genefragment, and comprised CDR3 loops varying in length between 6 and 12amino acids.

It is apparent that many modifications and variations of this inventionas hereinabove set forth may be made without departing from the spiritand scope thereof. The specific embodiments are given by way of exampleonly and the invention is limited only by the terms of the appendedclaims.

1. A method for obtaining a phage particle comprising an antibodyfragment directed against an antigen associated with the surface oftarget cells in a heterogeneous cell population, wherein saidheterogeneous cell population comprises non-target cells and targetcells in a heterogeneous mixture, and wherein said non-target cellsand/or target cells are detectably labeled, the method comprising: (a)providing a library of phage particles that express antibody fragmentson the surface of the phage particles; (b) incubating said library ofphage particles with said heterogeneous cell population under conditionsthat allow binding of the antibody fragment expressed on the surface ofthe phage particles to said antigen associated with said target cells;(c) separating said target cells and phage particles bound therewithfrom phage particles not bound to said target cells; and (d) recoveringthe phage particles bound to the target cells.
 2. A method according toclaim 1, wherein the separating of said target cells and phage particlesbound therewith from phage particles not bound to said target cells isaccomplished by flow cytometry.
 3. A method according to claim 1,further comprising isolating antibody fragments that bind to said targetcells.
 4. A method according to claim 1, wherein said detectably labeledcells are labeled with a fluorescent label.
 5. A method according toclaim 4, wherein said fluorescent label is a fluorescein label.
 6. Amethod according to claim 1, further comprising repeating steps (b)through (d).
 7. A method according to claim 1, wherein the library ofphage particles comprises phage particles expressing Fab or single chainFv (scFv) antibody fragments.